Microbial antibiotic resistance is spreading at an alarming pace, creating a situation which calls for new strategies to control microbes. In this context, approaches employing interference in the bacterial chemical communication (known as quorum sensing (QS)), that have the potential to control pathogens without killing commensal and health-associated bacteria, are extremely attractive. Numerous bacterial pathogens produce and utilize acyl homoserine lactones (AHLs) as chemical signal molecules to coordinate, in a cell density dependent manner, bacterial behaviors such as virulence and biofilm formation. Our lab has identified, characterized, and solved the structures of enzymatic quenchers of bacterial signaling, termed lactonases, which hydrolyze AHLs. The use of such Quorum Quenching (QQ) enzymes for pathogen control is fundamentally different from the use of antimicrobials: the enzymes show no toxicity, do not need to enter cells or bind to a receptor, but rather inhibit pathogenicity through signal disruption. We have demonstrated their striking ability to dramatically inhibit biofilm formation and bacterial virulence in vitro and in vivo. Yet, despite intensive efforts to characterize the effects of signal disruption, critical mechanistic questions remain, let alone the importance of signaling in the context of complex communities that remained inaccessible due to the lack of tools and methods. Excitingly, we have recently isolated, bioengineered and characterized enzymatic quenchers of bacterial signaling with exceptional catalytic and stability properties that unlock our ability to study the importance of QS in numerous contexts. We propose to take advantage of this new technology to investigate the importance of signaling in bacteria, at both the cellular and community levels. The scientific premise of our work is that controlled and effective signal disruption will lead to the mechanistic understanding of signaling at the cellular and community levels, including in communities relevant to disease and/or infection. Therefore, we will (i) explore the effects of signaling on pathogen-critical bacterial behaviors including virulence and biofilm formation for key lung pathogens; (ii) investigate the importance of signaling in mixed communities and the effect of its disruption on the microbial population and (iii) create the tools to study the specific importance of key signaling molecules at the cellular and community levels. This fundamental research will provide a critical understanding of signaling mechanisms in communities. As a result, it is expected to have broad impact on the field biology. Taking advantage of newly developed tools, it represents an opportunity to collect comprehensive and consistent insight at both the cellular and community levels of microbial signaling. Moreover, this research will establish the tools to resolve the specific contributions of the different types of AHLs used for signaling.